The overall objective of these studies is the understanding of the molecular basis of nutrient transport in mammalian cells. The transport of neutral amino acids into Chinese hamster ovary cells and Ehrlic ascites tumor cells is being studied. The specific aims include a determination of the properties of the transport systems and a study of the regulation of transport of cells grown in tissue culture. Genetic approaches are being developed to select for transport mutants of the Cinese hamster cells. Both transport activity and regulatory mutants will be isolated and characterized. Hybrid cells formed from human and Chinese hamster cells will be used to identify the chromosomal location of transport genes in both cell types. In addition, we are isolating and purifying transport components form Ehrlich ascites cell membranes. The purified transport components are used to reconstitute active transport in phospho-lipid vesicles. Methods being used to purify transport components include detergent solubilization of membranes and the development of antibodies to the solubilized membrane proteins. Procedures are being developed for cloning of the transport genes from mammalian cells. The understanding of membrane transport and its regulation will be important to the understanding of a number of human diseases which result from transport defects such as cystinuria and various other malabsorption syndromes.